A set of PCRs for rapid identification and characterization of Pseudomonas syringae phylogroups.

AIMS: The aim of this study was to develop a rapid PCR-based method for the specific detection of individual phylogroups of the Pseudomonas syringae complex. METHODS AND RESULTS: Seven primer pairs were developed by analysing whole genomes of 54 Ps. syringae strains. The specificity and sensitivity of these primer pairs were assessed on 236 strains from a large and comprehensive Ps. syringae collection. The method was also validated by characterizing the phylogenetic diversity of 174 putative Ps. syringae isolates from kiwifruit and apricot orchards of southeastern France. CONCLUSION: Our PCR-based method allows for the detection and characterization of nine of the 13 Ps. syringae phylogroups (phylogroups 1, 2, 3, 4, 7, 8, 9, 10 and 13). SIGNIFICANCE AND IMPACT OF THE STUDY: To date, phylogenetic affiliation within the Ps. syringae complex was only possible by sequencing housekeeping genes. Here, we propose a rapid PCR-based method for the detection of specific phylogroups of the Ps. syringae complex. Furthermore, for the first time we reveal the presence of Ps. syringae strains belonging to phylogroups 10 and 13 as epiphytes on plants, whereas they had previously only been observed in aquatic habitats.

A simple, rapid and efficient way to obtain infectious clones of potyviruses.

The availability of an infectious cDNA clone is a prerequisite for genetic studies on RNA viruses. However, despite important improvement in molecular biology techniques during the last decades, obtaining such clones often remains tedious, time-consuming and rather unpredictable. In the case of potyviruses, cDNA clones are frequently unstable due to the toxicity of some viral proteins for bacteria. The problem can be overcome by inserting introns into the viral sequence but this requires additional steps in the cloning process and depends on the availability of suitable restriction sites in the viral sequence or adjunction of such sites by mutagenesis. Homologous recombination in yeast rather than in vitro restriction and ligation can be used to build infectious clones or other viral constructs. This paper describes how, by using recombination in yeast and fusion PCR, infectious intron-containing clones were obtained within a few weeks for two strains of watermelon mosaic virus (WMV, Potyvirus), whereas previous attempts using "classical" cloning techniques had failed repeatedly. Using the same approach, intronless infectious clones of two other potyviruses, zucchini yellow mosaic virus (ZYMV) and papaya ringspot virus (PRSV), were obtained in less than two weeks.

Search for factors involved in the rapid shift in Watermelon mosaic virus (WMV) populations in South-eastern France.

Watermelon mosaic virus (WMV, genus Potyvirus, family Potyviridae) was reported for the first time in France in 1974, and it is now the most prevalent virus in cucurbit crops. In 2000, new strains referred as 'emerging' (EM) strains were detected in South-eastern France. EM strains are generally more severe and phylogenetically distinct from those previously reported in this country and referred as 'classic' (CL) strains. Since 2000, EM strains have been progressively replacing CL strains in several areas where they co-exist. In order to explain this rapid shift in virus populations, the biological properties of a set of 17 CL and EM WMV isolates were compared. No major differences were observed when comparing a limited host range including 48 different plant species or cultivars. Only two species were differential; Chenopodium quinoa was systemically infected by CL and not by EM isolates whereas Ranunculus sardous was systemically infected by EM and not by CL isolates. A considerable variability was observed in aphid transmission efficiencies but this could not be correlated to the CL or EM types. Two subsets of five isolates of each group were used to compare aphid transmission efficiencies from single and double (CL-EM) infections using six different cucurbit and non-cucurbit hosts. EM isolates were generally better transmitted from mixed CL-EM infections than CL isolates and CL transmission rates were significantly lower from double than from single infections. Cross-protection was only partial between CL and EM strains leading to frequent double infections, and only a slight asymmetry was observed in cross-protection efficiencies. Since double infections occur very commonly in fields, the preferential transmission of EM from mixed CL-EM infections could be one of the factors leading to the displacement of CL isolates by EM isolates.

Recombination in natural populations of watermelon mosaic virus: new agronomic threat or damp squib?

Since their introduction in south-eastern France around 1999, new, 'emerging' (EM) strains of watermelon mosaic virus (WMV) coexist with the 'classic' (CL) strains present for more than 40 years. This situation constitutes a unique opportunity to estimate the frequency of recombinants appearing in the few years following introduction of new strains of a plant RNA virus. Molecular analyses performed on more than 1000 isolates from epidemiological surveys (2004-2008) and from experimental plots (2009-2010), and targeting only recombinants that became predominant in at least one plant, revealed at least seven independent CL/EM or EM/EM recombination events. The frequency of recombinants involving at least one EM parent in the natural populations tested was on the order of 1 %. No new recombinant was detected for more than 1 year, and none but one in more than one location. In tests comparing host range and aphid transmissibility, the new recombinants did not display a better fitness than their 'parental' isolates. No recombinant was detected from artificial mixed infections of CL and EM isolates of various hosts after testing more than 1500 subcultures obtained after single-aphid transmission. These results constitute one of the first estimations of the frequency of recombinants in natural conditions for a plant RNA virus. This suggests that although viable recombinants of WMV are not rare, and although recombination may potentially lead to new highly damaging strains, the new recombinants observed so far had a lower fitness than the parental strains and did not emerge durably in the populations.

First Report of Zucchini yellow mosaic virus in Cucurbits in Ivory Coast.

During a field survey conducted in December 2008 and January 2009 in southern Ivory Coast, zucchini squash (Cucurbita pepo L.) and cucumber (Cucumis sativus L.) plants were observed showing severe symptoms of leaf mosaic and distortions, filiformism, and fruit deformations. Nine samples were collected from symptomatic plants in four locations (Adzopé, Songon, Ayamé, and Gagnoa) and dried over CaCl2. Double-antibody sandwich (DAS)-ELISA tests were performed directly on dried samples with antisera against nine cucurbit-infecting viruses: Zucchini yellow mosaic virus (ZYMV, Potyvirus); Papaya ringspot virus (PRSV, Potyvirus); Watermelon mosaic virus (WMV, Potyvirus); Moroccan watermelon mosaic virus (MWMV, Potyvirus); Cucumber vein yellowing virus (CVYV, Ipomovirus); Cucumber mosaic virus (CMV, Cucumovirus); Cucurbit aphid borne yellows virus (CABYV, Polerovirus); Squash mosaic virus (SqMV, Comovirus); and Cucumber green mottle mosaic virus (CGMMV, Tobamovirus). ZYMV was detected alone in four of six zucchini squash samples and in mixed infection with CMV and PRSV in two of three cucumber samples. A cucumber sample (CI09-09) collected at Songon and infected by ZYMV, CMV, and PRSV was inoculated to zucchini squash. ZYMV was separated from CMV and PRSV by inoculating zucchini squash plantlets with one Myzus persicae Sulzer per plant with 2-min acquisition and 2-h inoculation access periods. Plants infected by ZYMV only developed typical symptoms of severe mosaic, distortion, and filiformism on leaves. Total RNA was extracted from the original dried sample of CI09-09 using TRI-Reagent (Molecular Research Center Inc., Cincinnati, OH) (2). One-step reverse transcription (RT)-PCR was performed with our standard protocol and specific primers (2), yielding a 605-bp fragment corresponding to part of the polymerase (NIb) and coat protein (CP) coding regions. The nucleotide sequence of the NIb-CP fragment of Ivory Coast ZYMV isolate CI09-09 (GenBank No. HM450303) shared 98.5, 92.7, 80.5, and 75.7% identity with ZYMV isolates from France (isolate E9, HM641798), Florida (D13914), Singapore (AF014811), and Vietnam (DQ925449), respectively. Sequence comparison indicated that CI09-09 belongs to the phylogenetic cluster 1 of group A of ZYMV (2). ZYMV, first described in 1981, is now one of the most damaging viruses in cucurbit crops worldwide and is characterized by an important biological and molecular diversity (1,3). ZYMV has already been reported in several African countries, mostly in the northern and southern parts of the continent (1), but to our knowledge, this is the first report of ZYMV in Ivory Coast. Among African isolates, CI09-09 shared 97.5% identity with isolate Su06-22 from Sudan (HM641799) belonging to the phylogenetic cluster 1 of group A of ZYMV, 94 to 95% identity with isolates from neighboring Mali (HM005307-HM005312) belonging to cluster 2 of group A, and 79.6% identity with the divergent isolate R5A from Réunion Island (L29569) belonging to phylogenetic group B of ZYMV. The presence of ZYMV in four distant locations in southern Ivory Coast suggests that this virus constitutes a serious threat to cucurbit production in this country. References: (1) C. Desbiez and H. Lecoq. Plant Pathol. 46:809, 1997, (2) C. Desbiez et al. Virus Res. 85:5, 2002, (3) H. Lecoq et al. Virus Res. 141:190, 2009.

Emergence of new strains of Watermelon mosaic virus in South-eastern France: evidence for limited spread but rapid local population shift.

Severe symptoms caused by Watermelon mosaic virus (WMV) in zucchini squash leaves and fruits have been observed since 1999 in South-eastern (SE) France. Their appearance correlates with the introduction of new, "emerging" (EM) isolates distant at the molecular level from the "classic" (CL) isolates present for more than 30 years. To understand the origin and spread of EM isolates, a survey was performed between 2004 and 2007. WMV isolates collected were characterized by sequencing part of the polymerase and coat protein coding regions. This revealed the presence of EM isolates in SE France only, whereas CL isolates were widespread throughout the country. Besides, four subgroups of EM isolates were observed in SE France, suggesting multiple introductions. Recombinants between CL and EM groups, which probably arose locally, were observed during the survey. A strong geographic structure that remained stable during the 4 years was observed between different EM isolates. Our results showed that EM isolates did not spread over long distances, but rapidly replaced the pre-existing CL isolates in all sites where both groups occurred.

Molecular epidemiology of Zucchini yellow mosaic virus in France: an historical overview.

Cucurbit viruses are involved in complex and changing pathosystems in France, with new virus strains or species regularly reported. Zucchini yellow mosaic virus (ZYMV) is an archetypal emerging virus that was reported in France in 1979. It has since caused sporadic but occasionally very severe economic losses and its epidemiology still remains poorly understood. Partial sequencing of the viral genome has been used to characterize ZYMV isolates that occurred over a 29-year period in experimental plots at Montfavet, France (n=227), or that were received through a national survey for cucurbit viruses conducted in France from 2004 to 2007 (n=198). A total of 34 haplotypes were differentiated belonging to five molecular groups, three including isolates already described in France and two corresponding to isolates that emerged in France within the last 5 years. Comparison of haplotypes found at one location during successive years revealed contrasting situations. When they were either the same or closely related haplotypes, this suggested the availability of overwintering hosts, whereas when they belonged to different molecular groups this indicated shifts in viral populations with possible new introductions. The contribution of molecular epidemiology in tracing the origin of ZYMV in the French West Indies is also reviewed.